We have previously shown that Wnt5A can increase melanoma metastasis. Gene expression profiling analysis revealed that Wnt5A induction causes the downregulation of many genes. The few genes upregulated by Wnt5A include those involved in tumor cell metastasis such as CD44, and vimentin, involved in an epithelial to mesenchymal transition (EMT). Another gene upregulated upon Wnt5A overexpression was the gene p21. We have shown that p21 protein expression is also increased in a high wnt5a expressing melanoma cell line (M93) as compared to a low-expressing wnt5a expressing one (G361). Moreover, siRNA inhibition of Wnt5A resulted in a decrease in p21 expression in M93 cells. P21 activated kinase has been shown to increase melanoma motility, and Wnt5A can inhibit proliferation of several cell types. We hypothesize that decreases in cell proliferation may coincide with increases in metastatic ability, and are pursuing various mechanism by which to study this.[unreadable] [unreadable] Factors that induce the proliferation of melanoma cells include TGFbeta. Recent data from Hoek et al have shown that TGF-beta sensitive cells have higher proliferation rates, and are less metastatic. Those that are TGFbeta insensitive have lower proliferation rates, and are highly metastatic. Similarly, Wnt5A is expressed in this cohort, but not the other. Data from our laboratory indicates that indeed melanoma cells with more Wnt5A have increased expression of factors such as phospho-SMAD. What the link between tehee factors, WNt5A and melanoma progression may be is currently under investigation.[unreadable] [unreadable] Finally, we are interested in the effects of the aging microenvironment on tumor progression. Older individuals who present with melanoma have a worse prognosis, and this could be due to factors such as decreased immunity, or changes in the microenvironment. Klotho is a gene that interacts with Wnts, and whose knockdown can lead to accelerated aging. We are studying the relationship between klotho and Wnt5A using melanoma as a model, and normal, aging skin. We are in the process of determining whether older skin fibroblasts have an increase in Wnt5a, and a loss of klotho. If this is so, we will ask if we can make melanoma cells more invasive if we co-culture them with old vs young fibroblasts. Further, we will knock klotho out of the younger fibroblasts, and see what effects that has on secretion of different factors from these fibroblasts, and if we can accelerate senescence, and if those senescing fibroblasts make melanoma cells more invasive. On a long term basis we will also ask if oncogene induced senescence can influence surrounding cells in a co-culture system- eg, skin fibroblasts, and make them senesce and in turn, release factors that then cause tumor cells to invade. Gene expression profiling studies will also be performed to obtain a global overview of thes studies.